Bcecf am

Astrocyte cells (C6, rat astrocyte cell line) and endothelial cells (bEnd.3, mouse endothelial cell line) were from ATCC (Manassas, VA, USA). Cell culture inserts for 24-well plates (transparent PET membrane transwells, 0.4 µm pore size) were obtained from Dominique Dutscher (Alsace, France). Imaging Plate FC, 24-well plates, TC-surface was purchased from Ozyme (Saint Quentin Yvelines, France). The EVOM voltohmmeter system was from World Precision Instruments (Hertfordshire, UK). ZO-1 antibodies were from Life Technologies (cat 40-2200; Saint Aubin, France) and claudin-5 antibodies were from ABCAM (ab15106; Paris, France). P-gp and MRP-1 antibodies were from GeneTex (GTX23364; San Antonio, TX, USA) and Santa Cruz Biotechnology (sc-13960; Dallas, TX, USA), respectively. Standard protein and all compounds for Ringer HEPES buffer, BCECF-AM, probenicid, verapamil, sodium fluorescein (Na-Fl), were from Sigma-Aldrich (St. Quentin Fallavier, France). Rhodamine-123 was from Thermo Fisher Scientific (Eugene, OR, USA). Hydralazine, MTT kit and Dulbecco’s Modified Eagle’s Medium (DMEM) were also from Sigma-Aldrich. Antibodies and reagents for the detection of HIF-1α were products of R&D systems (Lille, France). YC-1 was purchased from VWR International (Fontenay-sous-Bois, France).

The analysis of pH_i was carried out in spermatozoa (30 × 10⁶ cells/mL) loaded with 5 μmol/L of the pH-sensitive dye BCECF-AM (B1150, Sigma-Aldrich®, Madrid, Spain) for 30 min at 38.5 °C. After that, the samples were centrifuged at 700×g for 3 min to remove the excess of dye and resuspended in PBS without Ca²⁺ and Mg²⁺ and incubated again for 15 min at 38.5 °C for the de-esterification of the dye. Finally, the samples were centrifuged and resuspended in NCAP, 0 mmol/L, 5 mmol/L, 15 mmol/L and 25 mmol/L of HCO₃⁻ for 1 and 60 min. The fluorescence was monitored using a spectrofluorometer (FP-6300, Jasco®, Cremella, Italy) every 2 s for a total time of 300 s. A calibration of the system was first performed using BCECF-AM stained and equilibrated spermatozoa at pH 6.0, 6.5, 7.0, 7.5 and 8.0 in the presence of 0.1% Triton X-100 by adjusting the pH with HCl and NaOH [27 (link)]. The emitted fluorescence ratio from the excitation at 490/440 nm was calculated and the regression line for extracellular pH (pH_e) vs. the 490/440 nm ratio was obtained (Additional file 1). The pH_i of sperm cells was estimated from the regression line.

Rabbit polyclonal antibody anti-AE1 was kindly provided by Dr C. Wagner (University of Zürich, Switzerland). Mouse monoclonal antibody anti-AE1 (BRIC6) came from the International Blood Group Reference Laboratory (IBGRL, Bristol, United Kingdom). Mouse monoclonal antibodies anti-ankyrin R (clone N388A/10) and anti-AQP1 (clone 1/A5F6) were from Neuromab (UC Davis/NIH, USA) and Bio-Rad (Marnes-la-Coquette, France) respectively, while goat polyclonal anti-stomatin (M-14) and rabbit polyclonal anti-CAII antibodies were purchased from Santa Cruz Biotechnology (Dallas, Texas USA). Secondary antibodies used in flow cytometry analysis were phycoerythrin (PE) or fluorescein isothiocyanate (FITC)-conjugated F(ab’)₂ fragment of goat anti-mouse and donkey anti-goat immunoglobulins from Beckman Coulter (Villepinte, France). BCECF-AM [2′,7′bis-(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester] and pyranine [8-hydroxypyrene-1,3,6-trisulfonic acid] fluorescent probes, as well as carbonic anhydrase from bovine erythrocytes and the AE1 inhibitor DIDS [4,4 2-Diisothiocyanatostilbene-2,2 2-disulfonic acid disodium salt] were purchased from Sigma-Aldrich (Saint Quentin, France). Chloride indicator SPQ (6-methoxy-N-(3-sulfopropy1) Quinolinium) was obtained from Invitrogen (Fisher Scientific, Illkirch, France).

Human blood was collected from healthy volunteers (Ethics number: REC 10/H0107/32) in 4.5 mL BD Vacutainer Citrate Tubes (Fisher Scientific; BD367691). Platelet-rich plasma (PRP) was obtained by centrifugation at 180 g for 17 min. PRP was isolated then supplemented with 0.02 U/mL apyrase, 140 nM prostaglandin E1 (PGE1; Sigma-Aldrich; P7527), and a 6:1 ratio of PRP to ACD (85 mM trisodium citrate, 71 mM citric acid, 111 mM dextrose). Platelet pellets were obtained by centrifugation at 520 g for 10 min and washed in CGS buffer (120 mM NaCl, 12.9 mM trisodium citrate, 30 mM dextrose, pH 6.5) supplemented with 0.02 U/mL apyrase and 140 nM prostaglandin E1. Platelets were centrifuged at 520 g for 10 min, resuspended in Tyrode’s buffer [10 mM HEPES, 145 mM NaCl, 3 mM KCl, 0.5 mM NaH₂PO₄, 1 mM MgSO₄, 0.1% (w/v) dextrose, pH 7.2] supplemented with 0.02 U/mL apyrase, 140 nM prostaglandin E1 and 4 μg/mL BCECF-AM (Sigma-Aldrich; B8806) and incubated for 30 min at room temperature. Platelets were centrifuged at 520 g for 10 min, adjusted to a concentration of 8 × 10⁷ platelets/mL in Tyrode’s buffer supplemented with 2 mM CaCl₂, 1 mM MgCl₂, and 0.5 U/mL bovine thrombin (Sigma-Aldrich; T4648), and incubated for 10 min at room temperature. To inactivate thrombin, platelets were then incubated with 2 U/mL hirudin for a further 10 min at room temperature.

To determine changes in the intracellular pH (pHi), pH sensitive cell permeabile fluorescent probe 2′,7′-biscarboxyethyl-5,6-carboxyfluorescein-acetoxymethyl ester (BCECF-AM; Sigma Aldrich) was used as described in Pastorek et al. [5 (link)]. Briefly, cells were loaded with 10 µmol/L BCECF and 0.5% pluronate in cultivation media without FBS for 45 min at 37 °C, 5 % CO₂, in dark. After subsequent washing with PBS buffer, fluorescence was measured at 490/535 nm and 440/535 nm on Synergy fluorescence scanner (BioTek, Bad Friedrichshall, Germany). The pHi signal was calibrated to bvpH0 by adding 10 µmol/L nigericin (Sigma Aldrich) with 130 mmol/L KCl. ΔpHi was ratiometric calculated from values obtained at 490 and 440/535 nm.

A total of 11 clinical strains used in this study were listed in Table 1. Tigecycline resistant K. pneumoniae clinical isolates were obtained from the CAMS Collection Center of Pathogen Microorganisms (CAMS-CCPM-A) in China. Tigecycline standard powder and N-Phenyl-1-naphthylamine (NPN) were purchased from TCI (Shanghai, China). Propidium iodide (PI) and ethidium bromide (EtBr) were purchased from Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China). Carbonyl cyanide m-chlorophenyl hydrazone (CCCP), 3,3’-dipropylthiadicarbocyanine iodide [DiSC₃(5)] and BCECF-AM were purchased from Sigma-Aldrich (St Louis, USA). An enhanced ATP Assay Kit and ROS Assay Kit were purchased from Beyotime Biotechnology (Shanghai, China). ML-7 hydrochloride, ML-9, HA-100, Ripasudil hydrochloride dihydrate, Fasudil hydrochloride, Hydroxyfasudil hydrochloride, tavaborole, and tegaserod maleate were purchased from Target Molecule Company (Boston, USA).

Brain slices were prepared in ice-cold high potassium solution containing (in mM): K-gluconate 120, HEPES acid 10, Na-gluconate 15, EGTA 0.2, NaCl 4 (pH 7.2, 290–300 mOsm). After cutting, slices were incubated in a high magnesium artificial cerebrospinal fluid (ACSF) containing (in mM): NaCl 125, KCl 2.5, CaCl₂ 0.8, MgCl₂ 8, NaHPO₄ 1.25, glucose 14, NaHCO₃ 24 (pH 7.3–7.4, 290–300 mOsm). Storage of slices and performing of experiments were conducted in ACSF, containing (in mM): NaCl 125, KCl 2.5, CaCl₂ 2.3, MgCl₂, 1.3, NaHPO₄ 1.25, glucose 14, NaHCO₃ 24 (pH 7.3–7.4, 290–300 mOsm). The ACSF was continuously oxygenated with 95% O₂ and 5% CO₂ to maintain the physiological pH. The following drugs were used: APV (40 μM, Hello Bio, Bristol, UK, Cat# HB0225), CNQX (10–40 μM, Sigma Aldrich, St. Quentin Fallavier, France, CAS: 115066-14-3), (-)-Bicuculline methochloride (10 μM or 40 μM, Tocris, Science Park Abingdon, UK, Cat#0131), BCECF/AM (10 μM, Sigma Aldrich, St. Quentin Fallavier, France, CAS: 117464-70-7). All drugs were diluted on the day of the experiment from the 1000× to 4000× stocks kept at −20 °C.